Method of separating ergosterol

ABSTRACT

The present invention provides a method of separating ergosterol from a solution containing ergosterol in water-insoluble organic solvent, comprising supplying water to the solution and precipitating ergosterol. According to the method of the present invention, ergosterol crystals can be obtained at a high yield. Moreover, controlled water supply provides granular ergosterol aggregates exhibiting good solid-liquid separation character at a high yield.

TECHNICAL FIELD

The present invention relates to a method of separating ergosterol.

BACKGROUND ART

Ergosterol is a type of sterols, which is contained in microorganismssuch as fungi. It is a substance useful as a provitamin D2. It has beenknown that when such an ergosterol is crystallized in a water-insolubleorganic solvent, granular aggregates exhibiting good solid-liquidseparation character can be obtained (Japanese Patent Laid-Open No.2002-80492). In this case, however, the actual yield of crystals issignificantly lower than that estimated from the solubility ofergosterol in the above organic solvent. Thus, this method isproblematic in that the remainder of ergosterol are graduallyprecipitated as fine crystalline flocculates from the filtrate aftersolid-liquid separation. It has also been known that ergosterol formshydrate crystals (S. E. Hull et al., Acta Cryst. B32, 2370-2373 (1976)).However, the association of this fact with the aforementioned problemhas not been clarified.

SUMMARY OF THE INVENTION

The present invention solves the aforementioned problem regarding pooryield, and provides an industrially advantageous method of separatingergosterol by obtaining ergosterol crystals at a high yield, andpreferably by obtaining ergosterol aggregates with good solid-liquidseparation character at a high yield.

As a result of various experiments and studies regarding crystallizationof ergosterol, the present inventor has found for the first time thatwater supply during the crystallization of ergosterol largely affectsthe yield of crystals. He has completed the present invention based onthese findings.

The method of separating ergosterol according to the present inventionis characterized in that water is supplied to a solution thereof in awater-insoluble organic solvent. According to the present invention,generation of ergosterol crystals is promoted, thereby achieving ahigher yield. In addition, by controlling the water supply, generationof ergosterol aggregates with good solid-liquid separation character ispromoted, thereby achieving high yield. Moreover, such ergosterolaggregates obtained by controlling water supply contains an amorphouscomponent in a crystal thereof, and has a crystallinity of approximately50% to 90%.

That is to say, the present invention relates to a method of separatingergosterol from a solution containing ergosterol in water-insolubleorganic solvent, which comprises supplying water to said solution andprecipitating ergosterol.

Moreover, the present invention relates to the above described method,wherein an amount of the water supplied is within such a range of amountthat no phase separation to form two liquid phases occurs between thewater-insoluble organic solvent and water.

Furthermore, the present invention relates to: the above describedmethod, wherein the solution containing ergosterol in thewater-insoluble organic solvent is a solution extracted from amicroorganism containing the ergosterol using the water-insolubleorganic solvent, or a solution obtained by extracting ergosterol fromthe microorganism using another solvent and then replacing said anothersolvent with the water-insoluble organic solvent;

the above described method, wherein the water-insoluble organic solventis hexane, heptane, octane, or a mixture thereof;

the above described method, wherein supplying water is conducted bycontinuously or intermittently moisturizing a gas phase portion withinan apparatus for precipitating ergosterol; and

the above described method, wherein ergosterol is separated byprecipitation as an aggregate having a crystallinity of 50% to 90%.

Still further, the present invention relates to an ergosterol aggregatehaving a crystallinity of 50% to 90%.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be described in detail below.

The method of separating ergosterol of the present invention ischaracterized in that it comprises supplying water to a solution thereofin a water-insoluble organic solvent containing ergosterol, so as toprecipitate ergosterol.

Ergosterol is a type of sterols, which is contained in microorganismssuch as fungi. It is a substance useful as a provitamin D2.Representative fungi containing ergosterol may include: mushrooms suchas Lentinus edodes or Grifola frondosa; yeasts; and leguminous bacteriafound in roots of leguminous plants. In addition, other microorganismscontaining ergosterol may include unicellular algae such as Chlorella.

The term “water-insoluble organic solvent” is used in the presentinvention to mean a substance that is generally considered to beinsoluble or hardly soluble in water based on Material Safety DataSheets, for example. Specific examples of such a water-insoluble organicsolvent may include: aliphatic hydrocarbons such as hexane, heptane, oroctane; aromatic hydrocarbons such as benzene, toluene, or xylene; andhalogenated hydrocarbons such as methylene chloride, chloroform, orcarbon tetrachloride. These solvents may be used singly or incombination of two or more types. In terms of necessity of conductingan. ergosterol precipitation operation, among the aforementionedsolvents, aliphatic hydrocarbons are preferable because the solubilityof ergosterol is low therein. Of these, hexane,'heptane, octane, and amixture thereof are more preferable.

Examples of a solution containing ergosterol in a water-insolubleorganic solvent may include: a solution thereof in a water-insolubleorganic solvent in which only ergosterol is dissolved; a solutionextracted from a microorganism containing the ergosterol, such as fungi,using a water-insoluble organic solvent; and a solution obtaining byextracting ergosterol from a microorganism containing ergosterol, suchas fungi, by using another solvent and then replacing said anothersolvent with the water-insoluble organic solvent.

Examples of another solvent used for extraction may include:water-soluble organic solvent such as acetone, ethanol, or 2-propanol;and mixtures comprising these water-soluble organic solvents and theaforementioned water-insoluble organic solvents.

When ergosterol is extracted from a microorganism containing theergosterol, such as fungi, using a water-insoluble organic solvent, theobtained extract may directly be used in the subsequent precipitationstep. Otherwise, after ergosterol has been extracted with awater-insoluble organic solvent (aromatic hydrocarbon or halogenatedhydrocarbon) in which the solubility of ergosterol is high, thewater-insoluble organic solvent may be exchanged with anotherwater-insoluble organic solvent (aliphatic hydrocarbon) in which thesolubility of ergosterol is low. The thus obtained solution may be usedin the subsequent precipitation step.

When ergosterol is precipitated from a solution thereof in awater-insoluble organic solvent containing the ergosterol, generallyknown methods can be applied. Examples of such known methods mayinclude: cooling crystallization of decreasing the temperature of asolution to decrease solubility, so as to precipitate a product ofinterest; and evaporation crystallization of evaporating an organicsolvent for concentration, so as to precipitate a product of interest.In the present invention, the cooling crystallization is applied morepreferably than the evaporation crystallization in which water isevaporated and lost by azeotropy or the like.

A system for precipitating ergosterol is not particularly limited.Examples of such a system may include: a batch crystallizer using anagitation tank equipped with a jacket; a continuous crystallizer forcontinuously supplying and discharging the liquid from one or moreagitation tanks equipped with a jacket; and a continuous towercrystallizer.

In the present invention, water is supplied to precipitate ergosterol.An amount of the water supplied is within such a range of amount that nophase separation to form two liquid phases occurs between thewater-insoluble organic solvent. The term “such a range of amount thatno phase separation to form two liquid phases occurs between thewater-insoluble organic solvent” is used herein to mean a trace amountof water that can be dissolved in a water-insoluble organic solvent.Such an amount of water is associated with water solubility in thewater-insoluble organic solvent, and thus, it varies somewhat dependingon the type of a water-insoluble organic solvent. When hexane is used assuch a water-insoluble organic solvent, for example, the amount of wateris preferably approximately 1 to 100 ppm with respect to the hexane.

If the amount of water is too large, the water-insoluble organic solventsolution is separated into two liquid phases, a water-insoluble organicsolvent phase and water phase. As a result, it becomes difficult tohandle, and also ergosterol appears as needle crystals, therebyresulting in a rather poorer solid-liquid separation character. Incontrast, if no water is supplied, crystals are not precipitated, andthe yield thereby decreases.

If water is supplied in a liquid form, even within a range where thesolution is not separated into two liquid phases, the liquid phasetransiently may become nonuniform, and needle crystals may then beformed at a position where water is localized. In order to prevent sucha phenomenon, it is preferable that water is supplied by moisturizing agas phase portion (a portion where the gas exists) in a system forprecipitating ergosterol, so as to maintain the liquid phase in auniform state.

A method of moisturizing a gas phase portion is not particularlylimited, as long as it can supply water to such an extent that thewater-insoluble organic solvent solution is not separated into twoliquid phases. Examples of such a method may include: direct feeding ofsteam; flowing of nitrogen gas or the like that has passed throughwater; supply of misty water generated using an ultrasonic humidifier;and flowing of a gas that has passed though the misty water as generatedabove.

Moreover, it is preferable that water is supplied continuously orintermittently. More preferably, water is supplied by continuously orintermittently moisturizing a gas phase portion of a system forprecipitating ergosterol.

A crystallization temperature is preferably −30° C. to 80° C., and morepreferably −20° C. to 60° C. A crystallization time is preferably 0.5 to24 hours, and more preferably 1 to 6 hours. When crystallization iscarried out by gradual cooling, a cooling rate is preferably 0.05° C. to3° C./min, and more preferably 0.1° C. to 1° C./min.

After ergosterol has been precipitated as described above, solid-liquidseparation is conducted by filtration or the like, so as to separateergosterol. Thereafter, the separated ergosterol is dried at ordinarytemperature or while warming, under ordinary or reduced pressure, so asto obtain ergosterol crystals.

The recovery rate of ergosterol is preferably 60% or more, morepreferably 70% or more, further preferably 80% or more, and particularlypreferably 90% or more.

Ergosterol precipitated and separated by particularly controlling thewater supply in the method of separating ergosterol of the presentinvention is a granular ergosterol aggregate with good solid-liquidseparation character. This aggregate does not only comprise generallyknown hydrate crystals, but also comprises amorphous component as wellas hydrate crystals in the crystals thereof. Since such amorphouscomponent contain no crystal water, an aggregate can efficiently beprecipitated with water supply that are smaller than those necessary forobtaining the aggregate entirely in the form of hydrate crystals,thereby improving the yield. Thus, it is preferable that the amount ofsuch amorphous component is larger. However, if the amount is too large,the resultant product cannot be obtained in the form of an aggregate.Accordingly, the crystallinity of an ergosterol aggregate is preferably50% to 90%, and more preferably 60% to 80%.

Since an aggregate with such characteristics still requires a minuteamount of water to constitute crystal component, if no water is suppliedduring the precipitation of ergosterol, water contained in the systemwill be consumed up, so that the yield cannot be increased.

The aforementioned crystallinity can be measured by X-ray diffraction orthe like. Moreover, it can also be obtained by measuring water ofhydration by thermogravimetric analysis. Herein, the value obtained bymeasuring by thermogravimetric analysis is adopted as a crystallinity.That is, an aggregate that has been dried by air so as not to losecrystal water is subjected to thermogravimetric analysis, so that thewater content thereof is measured, thereby obtaining the amount ofcrystal component in the aggregate, that is, a crystallinity.

Specifically, such a crystallinity can be obtained as follows. It isconsidered that the crystal component in the air-dried aggregatecorrespond to a monohydrate and that the amorphous component contains nowater. Thus, the number of moles of water present in the aggregate=thenumber of moles of crystals. In addition, when the weight of a samplesubjected to the analysis is expressed as W and the loss weightmeasurement is expressed as Δw, the following formulas hold: The numberof moles of crystals (Mc)=Δw/the molecular weight of water; and

The total number of moles of ergosterol (M)=(W−Δw)/the molecular weightof ergosterol. Thus, a crystallinity is obtained by the followingformula:

Crystallinity=(Mc/M)×100.

The particle size of the obtained granular aggregate is preferably 50 μmor greater, and more preferably 100 μm or greater.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be further described in the followingexamples. However, the examples are not intended to limit the scope ofthe present invention.

Example 1

500 ml of a hexane solution containing ergosterol with a concentrationof 4 g/L was filtrated and then placed in a batch crystallizer equippedwith a 500 ml separable flask. While stirring, the solution was cooledat a cooling rate of 0.2° C./min until the liquid temperature decreasedfrom 45° C. to 10° C. After the liquid temperature became 35° C., theupper portion of the flask was opened every 25 minutes, so that using asyringe, 100 ml each of the air moisturized with hot water at 40° C. wasfed into the flask. After completion of the cooling crystallization, thesolid was separated from the liquid using a vacuum filtration system,and then the solid was dried, so as to obtain an aggregate. The thusobtained crystal was a granular aggregate having a particle size between100 μm and 200 μm. The granular aggregate exhibited good solid-liquidseparation character, and the recovery rate (obtained by measuring theconcentration of the solution using the additive amount as a standard)was also favorable, resulting in 78%. In addition, the reduced weightwas measured by increasing the temperature from 30° C. to 180° C. at arate of 10° C./min, using a thermogravimetric system (TG/DTA220;manufactured by Seiko Instruments Inc.), so as to obtain acrystallinity. As a result, the crystallinity of this aggregate wasfound to be 66%.

Example 2

A hexane solution containing ergosterol whose concentration had beenadjusted to be 4 g/L was continuously supplied to a continuous towercrystallizer having an inside diameter of 30 mm and a height of 450 mm.The crystallizer was operated under conditions consisting of: thetemperature of a discharging port at the head of the tower of 10° C.; arotation rate by stirring of 100 rpm; a liquid-supplying rate of 5ml/min (wherein the supplied liquid was maintained at approximately 50°C.) ; and a mean retention time of 60 minutes. A nitrogen gas, which wasmoisturized by passing it through a trap tube containing water, wasflown through a gas phase portion from a nozzle located at the top endof the above system. Crystals precipitated and sedimented in the systemwere sampled. As a result, it was found that the crystal was a granularaggregate with a particle size of approximately 200 μm, which exhibitedgood solid-liquid separation character. After completion of theoperation for 1 hour, the recovery rate (obtained by measuring theconcentration of the. solution existing the exit of the system, usingthe additive amount as a standard) was favorable, resulting in 77%.Moreover, thermogravimetric analysis was carried out in the same abovemanner. As a result, this crystal was found to have a crystallinity of60%.

Example 3

Hexane solution (500 ml) containing ergosterol with a concentration of 4g/L was filtrated and then added to a batch crystallizer of a 500 mlseparable flask. While stirring, the solution was cooled at a coolingrate of 0.2° C./min until the liquid temperature decreased from 45° C.to 10° C. During the cooling, 0.5 ml of water was added. The added watercould not be dissolved in hexane, and existed as a droplet untilcompletion of the cooling. After completion of the cooling, the solidwas separated from the liquid, and a crystal was collected after dryingit under reduced pressure. The recovery rate (obtained by measuring theconcentration of the solution using the additive amount as a standard)was favorable, resulting in 87%. However, the crystal had a needle form.Moreover, thermogravimetric analysis was carried out in the same abovemanner. As a result, this crystal was found to have a crystallinity of95%.

Comparative Example 1

A hexane solution (500 ml) containing ergosterol with a concentration of4 g/L was filtrated and then added to a batch crystallizer of a 500 mlseparable flask. While stirring, the solution was cooled at a coolingrate of 0.2° C./min until the liquid temperature decreased from 45° C.to 10° C. The system was hermetically sealed until the cooling operationwas completed. After completion of the cooling, the solid was separatedfrom the liquid, and a crystal was collected after drying it underreduced pressure. The obtained crystal was a granular aggregate having aparticle size of approximately 200 μm, which exhibited good solid-liquidseparation character. However, the recovery rate (obtained by measuringthe concentration of the solution. using the additive amount as astandard) was poor, resulting in 53%.

INDUSTRIAL APPLICABILITY

The present invention makes it possible to obtain ergosterol crystals ata high yield. Moreover, by controlling the water supply, it becomespossible to obtain a granular ergosterol aggregate exhibiting goodsolid-liquid separation character at a high yield.

1-7. (canceled)
 8. A method of separating ergosterol from a solutioncontaining ergosterol in an organic solvent, which comprises: supplyingan amount of water to a solution containing ergosterol in an organicsolvent that is an aliphatic hydrocarbon, aromatic hydrocarbon,halogenated hydrocarbon or a mixture thereof, and precipitatingergosterol from said solution by cooling crystallization, wherein theamount of water supplied is within the range of water solubility in saidorganic solvent.
 9. The method according to claim 8, wherein saidsolution containing ergosterol in an organic solvent is obtained byextracting ergosterol from a microorganism containing ergosterol usingsaid organic solvent, or is obtained by extracting ergosterol from amicroorganism containing ergosterol using a second solvent that isdifferent from said organic solvent, and then replacing said secondsolvent with said organic solvent.
 10. The method according to claim 8,wherein said organic solvent is a hexane, a heptane, an octane, or amixture thereof.
 11. The method according to claim 8, wherein theergosterol is separated by precipitation as an aggregate comprising anamorphous component as well as hydrate crystals in the crystals thereof.12. The method according to claim 11, wherein the ergosterol isseparated by precipitation as an aggregate having a crystallinity of 50%to 90%, wherein crystallinity is the amount of crystal component in theaggregate as determined by measuring water of hydration bythermogravimetric analysis.
 13. The method according to claim 8,wherein: said organic solvent is an aliphatic hydrocarbon; and whereinsaid solution containing ergosterol in an organic solvent is obtained byextracting ergosterol from a microorganism containing ergosterol using asecond solvent that is an aromatic hydrocarbon or a halogenatedhydrocarbon, and then replacing said second solvent with said organicsolvent.
 14. A method of separating ergosterol from a solutioncontaining ergosterol in an organic solvent, which comprises: supplyingan amount of water to a solution containing ergosterol in an organicsolvent that is an aliphatic hydrocarbon, aromatic hydrocarbon,halogenated hydrocarbon or a mixture thereof, and precipitatingergosterol from said solution by cooling crystallization, wherein theamount of water is supplied within such a range of amount that no phaseseparation to form two liquid phases occurs between said organic solventand water.
 15. The method according to claim 14, wherein said solutioncontaining ergosterol in an organic solvent is obtained by extractingergosterol from a microorganism containing ergosterol using said organicsolvent, or is obtained by extracting ergosterol from a microorganismcontaining ergosterol using a second solvent that is different from saidorganic solvent, and then replacing said second solvent with saidorganic solvent.
 16. The method according to claim 14, wherein saidorganic solvent is a hexane, a heptane, an octane, or a mixture thereof.17. The method according to claim 14, wherein the ergosterol isseparated by precipitation as an aggregate comprising an amorphouscomponent as well as hydrate crystals in the crystals thereof.
 18. Themethod according to claim 17, wherein the ergosterol is separated byprecipitation as an aggregate having a crystallinity of 50% to 90%,wherein crystallinity is the amount of crystal component in theaggregate as determined by measuring water of hydration bythermogravimetric analysis.
 19. The method according to claim 14,wherein: said organic solvent is an aliphatic hydrocarbon; and whereinsaid solution containing ergosterol in an organic solvent is obtained byextracting ergosterol from a microorganism containing ergosterol using asecond solvent that is an aromatic hydrocarbon or a halogenatedhydrocarbon, and then replacing said second solvent with said organicsolvent.